The detection of enzymatic activity is important in many aspects of biological research and in medical diagnosis and treatment. Abnormal enzymatic activity can be the cause of a disease state and/or a diagnostic and/or prognostic marker for a particular condition. There is a need in the art for improved assays for enzymatic activity such as protease activity.
For example, factor Xa (FXa), an activated serine endopeptidase, is a common blood factor involved in multiple coagulation pathways. Measurement of FXa activity is commonly employed in the determination of the activity of upstream coagulation factors including factor VIII (FVIII) and factor IX (FIX), as well as in the determination of the anticoagulant properties of pharmaceutical compounds such as heparin. Commercially available kits for these purposes typically contain chromogenic based substrates containing the amino acid sequence IEGR (SEQ ID NO:1), which is recognized and cleaved by FXa, resulting in the severing of the peptide bond between the arginine residue and the next amino acid or molecule toward the carboxy-terminus. For example, substrate S2222, Bz-Ile-Glu[γ-OR]-Gly-Arg-pNA.HCl (Para-Nitro-Aniline) [R═H (50%) and R═CH3 (50%)], a polypeptide with a pNA conjugated at its C-terminus, is found in several colorimetric kits for FVIII and FIX detection. FXa can digest this substrate and release pNA which, in its free form, produces a bright yellow color which can be quantitatively measured in an absorbance reader. Commercially available FVIII detection kits include BIOPHEN Factor IX (Ref. A221802. Aniara, Mason, Ohio 45040), BIOPHEN FVIII:C (Ref. A221406. Aniara), CHROMOGENIX COATEST® SP4 Factor VIII (Cat. K824094. DiaPharma Group, Inc. West Chester, Ohio 45069) and Rox Factor IX (Cat. 900020. DiaPharma Group, Inc.). An assay for detecting FVIII activity is described in U.S. Pat. No. 5,506,112.
Additionally, caspase enzymes are proteases involved in cellular inflammation and apoptotic cascades. The assessment of caspase enzymatic activity can be used to evaluate cell death pathways and new apoptosis-modulating agents. Failure of apoptosis has been implicated as one of the causes of many different diseases including tumor development and autoimmune diseases. Additionally, undesirable apoptosis can occur with ischemia or Alzheimer's disease.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.